高产纤维素酶菌株的筛选、鉴定及产酶特性研究
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摘要
为了从腐殖质土壤中筛选得到高产纤维素酶菌株,本试验进行了产纤维素酶菌株的筛选、鉴定及其产酶特性研究。首先用羧甲基纤维素钠(CMA-Na)培养基初步分离产纤维素酶菌株,再经过酶活复筛得到一株高产纤维素酶菌株B17。经过形态学和16S rDNA测序鉴定为枯草芽孢杆菌(Bacillus subtilis),并对其发酵产酶条件和酶学性质进行初步研究。试验结果表明,菌株B17的最佳发酵条件为:培养时间3 d、发酵温度35℃、初始pH为6.0,该条件下测得CMC酶活达85.48 U/mL、FPA酶活达59.85 U/mL。酶学性质研究表明:菌株所产纤维素酶最适反应条件为温度50℃、pH为6.0,且具在30～60℃、pH为4.0～7.0范围均具有较高酶活。以上结果表明,本研究所得菌株B17具有较高的开发价值,可应用于农作物秸秆饲料的生产。
In order to isolate cellulose-producing strain from soil samples of decayed leaf,the screening and identification of a cellulase-producing bacterium and its cellulase characterization were measured in this study.Firstly,the cellulose-producing strain was preliminarily isolated by carboxymethyl cellulose sodium(CMA-Na) culture medium,and then a high-yield cellulase strain B17 was obtained by enzymatic rescreening.Based on morphological observation and 16 S rDNA sequencing,B17 was identified as Bacillus subtilis,and then it's fermentation conditions and enzymatic properties were preliminarily explored.The results showed that the optimal fermentation conditions for the cellulose-producting strain B17 were that the cultivate time,temperature and pH were 3 d,35 ℃ and 6.0.And under this condition,the maximum CMC activity and FPA activity were 85.48 and 59.85 U/mL,respectively.According to the results of enzymatic properties,the optimal temperature and pH for the enzyme were 50 ℃ and 6.0. Enzyme stability studies showed that enzyme kept active when the temperature was 30 to 60 ℃ and pH ranged from 4.0 to 7.0.In conclusion,a Bacillus subtilis with high cellulose activity had been isolated and identified,and it had high value for the feed's production of crop straw.
In order to isolate cellulose-producing strain from soil samples of decayed leaf,the screening and identification of a cellulase-producing bacterium and its cellulase characterization were measured in this study.Firstly,the cellulose-producing strain was preliminarily isolated by carboxymethyl cellulose sodium(CMA-Na) culture medium,and then a high-yield cellulase strain B17 was obtained by enzymatic rescreening.Based on morphological observation and 16 S rDNA sequencing,B17 was identified as Bacillus subtilis,and then it's fermentation conditions and enzymatic properties were preliminarily explored.The results showed that the optimal fermentation conditions for the cellulose-producting strain B17 were that the cultivate time,temperature and pH were 3 d,35 ℃ and 6.0.And under this condition,the maximum CMC activity and FPA activity were 85.48 and 59.85 U/mL,respectively.According to the results of enzymatic properties,the optimal temperature and pH for the enzyme were 50 ℃ and 6.0. Enzyme stability studies showed that enzyme kept active when the temperature was 30 to 60 ℃ and pH ranged from 4.0 to 7.0.In conclusion,a Bacillus subtilis with high cellulose activity had been isolated and identified,and it had high value for the feed's production of crop straw.
引文
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[17] 陆晨.高产纤维素酶菌株的筛选及其产酶条件的优化[D].株洲:中南林业科技大学,2012. LU C.Screening and identification of efficient cellulase-producing fungus and optimization of the fermentation condition[D].Zhuzhou:Central South University of Forestry and Technology,2012.(in Chinese)
[18] 王加友,赵彭年,杨德玉,等.一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果[J].生物技术进展,2018,8(2):132-139. WANG J Y,ZHAO P N,YANG D Y,et al.Screening and identification of a cellulose decomposing fungus and its degradation effect on corn straw[J].Current Biotechnology,2018,8(2):132-139.(in Chinese)
[19] 张玲秀,白建华,郝瑞林,等.一株粗糙脉胞菌的鉴定及产纤维素酶发酵工艺优化[J].中国饲料,2018,1:27-31. ZHANG X L,BAI J H,HAO R L,et al.A strains of Neurospora crassa identification and produce cellulose enzyme fermentation process optimization[J].Chinese Feed,2018,1:27-31.(in Chinese)
[20] 明红梅,邹伟,陈晓旭,等.白酒糟高温纤维素分解菌的筛选、产酶分析与鉴定[J].饲料研究,2015,11:24-27. MING H M,ZOU W,CHEN X X,et al.Screening and identification of cellulolytic bacteria in liquor lees with high temperature[J].Feed Research,2015,11:24-27.(in Chinese)
[21] 柴秀娟,李曹龙,孔德真,等.产纤维素酶菌株的筛选、鉴定及产酶条件的优化[J].生物技术通报,2014,9:164-170. CHAI X J,LI C L,KONG D Z,et al.Screening,identification of a cellulase-producing bacterium strain and its enzyme-producing conditions[J].Biotechnology Bulletin,2014,9:164-170.(in Chinese)
[22] 韦海婷,刘晗璐,李光玉,等.梅花鹿瘤胃纤维素降解菌的分离鉴定及其酶活力测定[J].中国畜牧兽医,2018,45(4):888-897. WEI H T,LIU H L,LI G Y,et al.Isolation,identification and determination of cellulose-degrading bacteria from Sika deer (Cervus nippon)[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):888-897.(in Chinese)
[23] 马军,王耀嵘,侯萍,等.一株源于红树林根际土壤产纤维素酶芽孢杆菌的鉴定及其酶学性质分析[J].海洋与湖沼,2016,47(5):997-1004. MA J,WANG Y R,HOU P,et al.Dentification and enzymatic property of cellulase-producing Bacillus subtilis isolated from mangrove rhizosphere soil[J].Oceanologia et Limnologia Sinica,2016,47(5):997-1004.(in Chinese)
[2] ZHENG Y,ZHANG S,MIAO S,et al.Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass[J].Journal of Biotechnology,2013,166(3):135-143.
[3] MOKATSE K M P,WYK J P H V.Relative saccharification of various waste paper materials by cellulase from Aspergillus niger[J].Development,2015,3:44-46.
[4] 柴秀娟.高产纤维素酶菌株的筛选、酶学性质及发酵工艺的研究[D].石河子:石河子大学,2015. CAI X J.Screening,enzyme-producing conditions optimized and enzymatic characteristics of cellulose-producing strain with high cellulase activity[D].Shihezi:Shihezi University,2015.(in Chinese)
[5] 田云,曹林友,周赓,等.一株纤维素酶高产菌的筛选、鉴定与产酶研究[J].化学与生物工程,2016,33(7):34-39. TIAN Y,CAO L Y,ZHOU G,et al.Screening,identification and enzyme-producing study of a high cellulase-producing strain[J].Chemistry and Bioengineering,2016,33(7):34-39.(in Chinese)
[6] GUPTA P,SAMANT K,SAHU A.Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential[J].International Journal of Microbiology,2012,2012(6):578925.
[7] CHIMTONG S,TACHAAPAIKOON C,PASON P,et al.Isolation and characterization of endocellulase-free multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1[J].Journal of Microbiology & Biotechnology,2011,21(3):284-392.
[8] KAUR J,CHADHA B S,SAINI H S.Regulation of cellulase production in two thermophilic fungi Melanocarpus sp.MTCC3922 and Scytalidium thermophilum MTCC4520[J] Enzyme & Microbial Technology,2006,38(7):931-936.
[9] ZHANG Q,LO C M,JU L K.Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30[J].Bioresource Technology,2007,98(4):753-760.
[10] 王洪媛,范丙全.三株高效秸秆纤维素降解真菌的筛选及其降解效果[J].微生物学报,2010,50(7):870-875. WANG H Y,FAN B Q.Screening of three straw-cellulose degrading microorganis[J].Acta Microbiologica Sinica,2010,50(7):870-875.(in Chinese)
[11] 李碧婵,陈金燕,苏雅娜,等.纤维素酶高产细菌的筛选、鉴定及酶学特性分析[J].湖北农业科学,2016,55(17):4572-4576. LI B C,CHEN J Y,SU Y N,et al.Screening and identification of a cellulase producing bacterium and its cellulase characterization[J].Hubei Agricultural Sciences,2016,55(17):4572-4576.(in Chinese)
[12] 江国忠.高产纤维素酶枯草芽孢杆菌的筛选、应用及其产酶条件研究[D].南昌:南昌大学,2010. JIANG G Z.Screening,application and conditions of enzyme production research of Bacillus subtilis strains with high-cellulase yield[D].Nanchang:Nanchang University,2010.(in Chinese)
[13] SWAIN M R,RAY R C.Biocontrol and other beneficial activities of Bacillus subtilis isolated from cowdung microflora[J]. Microbiological Research,2009,164(2):121-130.
[14] WALSH C.Enabling the chemistry of life[J].Nature,2001,409(6817):226-231.
[15] RIVEDI N,GUPTA V,KUMAR M,et al.An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca[J].Carbohydrate Polymers,2011,83(2):891-897.
[16] 李洋.纤维素酶产生菌的筛选及产酶条件优化[D].大连:大连理工大学,2015. LI Y.Isolation of a cellulase-producing fungus and optimization of fermentation condition for enzyme production[D].Dalian:Dalian University of Technology,2015.(in Chinese)
[17] 陆晨.高产纤维素酶菌株的筛选及其产酶条件的优化[D].株洲:中南林业科技大学,2012. LU C.Screening and identification of efficient cellulase-producing fungus and optimization of the fermentation condition[D].Zhuzhou:Central South University of Forestry and Technology,2012.(in Chinese)
[18] 王加友,赵彭年,杨德玉,等.一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果[J].生物技术进展,2018,8(2):132-139. WANG J Y,ZHAO P N,YANG D Y,et al.Screening and identification of a cellulose decomposing fungus and its degradation effect on corn straw[J].Current Biotechnology,2018,8(2):132-139.(in Chinese)
[19] 张玲秀,白建华,郝瑞林,等.一株粗糙脉胞菌的鉴定及产纤维素酶发酵工艺优化[J].中国饲料,2018,1:27-31. ZHANG X L,BAI J H,HAO R L,et al.A strains of Neurospora crassa identification and produce cellulose enzyme fermentation process optimization[J].Chinese Feed,2018,1:27-31.(in Chinese)
[20] 明红梅,邹伟,陈晓旭,等.白酒糟高温纤维素分解菌的筛选、产酶分析与鉴定[J].饲料研究,2015,11:24-27. MING H M,ZOU W,CHEN X X,et al.Screening and identification of cellulolytic bacteria in liquor lees with high temperature[J].Feed Research,2015,11:24-27.(in Chinese)
[21] 柴秀娟,李曹龙,孔德真,等.产纤维素酶菌株的筛选、鉴定及产酶条件的优化[J].生物技术通报,2014,9:164-170. CHAI X J,LI C L,KONG D Z,et al.Screening,identification of a cellulase-producing bacterium strain and its enzyme-producing conditions[J].Biotechnology Bulletin,2014,9:164-170.(in Chinese)
[22] 韦海婷,刘晗璐,李光玉,等.梅花鹿瘤胃纤维素降解菌的分离鉴定及其酶活力测定[J].中国畜牧兽医,2018,45(4):888-897. WEI H T,LIU H L,LI G Y,et al.Isolation,identification and determination of cellulose-degrading bacteria from Sika deer (Cervus nippon)[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):888-897.(in Chinese)
[23] 马军,王耀嵘,侯萍,等.一株源于红树林根际土壤产纤维素酶芽孢杆菌的鉴定及其酶学性质分析[J].海洋与湖沼,2016,47(5):997-1004. MA J,WANG Y R,HOU P,et al.Dentification and enzymatic property of cellulase-producing Bacillus subtilis isolated from mangrove rhizosphere soil[J].Oceanologia et Limnologia Sinica,2016,47(5):997-1004.(in Chinese)
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